Food Analytical Methods

, Volume 6, Issue 3, pp 715–726 | Cite as

TLC–Bioautography-Guided Isolation, HPTLC and GC–MS-Assisted Analysis of Bioactives of Piper betle Leaf Extract Obtained from Various Extraction Techniques: In vitro Evaluation of Phenolic Content, Antioxidant and Antimicrobial Activities

  • H. V. Annegowda
  • P. Y. Tan
  • M. N. Mordi
  • S. Ramanathan
  • M. R. Hamdan
  • M. H. Sulaiman
  • S. M. Mansor


The polyphenolic content, antioxidant and antibacterial activities of the ethanolic extracts of Piper betle leaf obtained from soxhlet (PBSx), sonication (PBSn) and maceration (PBMn) extraction methods were studied. GC–MS analysis was carried out to determine the variation in the phytoconstituents in these extracts. Whereas, thin-layer chromatography (TLC)–bioautography was conducted to localise, separate, and identify antioxidants, and their amount was determined by the newly developed high-performance thin-layer chromatography (HPTLC) method. The results of polyphenolic content, antioxidant assays and antimicrobial assay showed that PBSn contained significant amount of polyphenolics, antioxidant and antimicrobial activity followed by PBMn and PBSx. Moreover, the obtained antioxidant activity of PBSn was significant even in comparison with butylated hydroxytoluene (BHT) and commercially available grape seed extract (GRSx). In addition, GC–MS analysis shown marked variations in the amount of the phytoconstituents among all these extracts with PBSn containing higher amount followed by PBMn and PBSx. TLC bioautography resulted in the separation of three compounds which are identified as eugenol, allylpyrocatechol, and eugenyl acetate. The HPTLC densitometric determination was also supported the results of antioxidant assays by revealing the presence of higher amount of identified antioxidants in PBSn followed by PBMn and PBSx. Since, P. betle leaf extract has been used as one of the ingredients in several herbal formulations, results of this study will not only help the herbal industries in choosing the appropriate extraction technique but also the developed HPTLC method was simple, precise, sensitive and accurate hence can be utilised for the routine quality control and standardisation of formulations containing P. betle leaf extract.


P. betle Sonication TLC–bioautography HPTLC validation Eugenol Allylpyrocatechol 



This project was funded by USM Research University Grant, Malaysia. The author (Annegowda H.V.) gratefully acknowledges the Institute of Postgraduate Studies of USM, Malaysia for granting USM Fellowship.


  1. Abou-Donia AH, Toaima SM, Hammoda HM, Shawky E (2008) New rapid validated HPTLC method for the determination of galanthamine in Amarallidaceae plant extracts. Phytochem Anal 19:353–358CrossRefGoogle Scholar
  2. Annegowda HV, Mordi MN, Ramanathan S, Hamdan MR, Mansor SM (2012) Effect of extraction techniques on phenolic content, antioxidant and antimicrobial activity of Bauhinia purpurea: HPTLC determination of antioxidants. Food Anal Methods 5:226–223CrossRefGoogle Scholar
  3. Arambewela LSR, Arawwawala LDAM, Ratnasooriya WD (2005a) Anti-diabetic activities of aqueous and ethanolic extracts of Piper betle leaves in rats. J Ethnopharmacol 102:239–245CrossRefGoogle Scholar
  4. Arambewela LSR, Arawwawala LDAM, Ratnasooriya WD (2005b) Antinociceptive activities of aqueous and ethanol extracts of Piper betle leaves in rats. Pharm Biol 43(9):766–772CrossRefGoogle Scholar
  5. Bauer AW, Kirby WMM, Sherris JC, Truck M (1966) Antibiotic susceptibility testing by a standardised single disc method. Am J Clin Pathol 45:493–496Google Scholar
  6. Benzie IFF, Strain JJ (1996) The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power, the FRAP assay. Anal Biochem 239:70–76CrossRefGoogle Scholar
  7. Blois MS (1958) Antioxidant determinations by the use of a stable free radical. Nature 181:1199–1200CrossRefGoogle Scholar
  8. Choudhary D, Kale RK (2002) Antioxidant and non-toxic properties of Piper betle leaf extract: in vitro and in vivo studies. Phytother Res 16:461–466CrossRefGoogle Scholar
  9. Elof JN (1998) A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica 64:711–713CrossRefGoogle Scholar
  10. Gallo FR, Multari G, Giambenedetti M, Federici E (2008) Chemical fingerprinting of Lawsonia inermis L. using HPLC, HPTLC and densitometry. Phytochem Anal 19:550–559CrossRefGoogle Scholar
  11. Gu L, Wu T, Wang Z (2009) TLC bioautography-guided isolation of antioxidants from fruit of Perilla frutescens var. acuta. LWT—Food Sci Technol 42:131–136Google Scholar
  12. Jantan I, Ahmad AR, Ahmad AS, Ali AM (1994) A comparative study of the essential oils of five Piper species from Peninsular Malaysia. Flavour Frag J 9:339–342CrossRefGoogle Scholar
  13. Jeng JH, Chen SY, Liao CH, Tung YY, Lin BR, Hahn LJ, Chang MH (2002) Modulation of platelet aggregation by areca nut and betle leaf ingredients: roles of reactive oxygen species and cyclooxygenase. Free Radic Biol Medicine 32:860–871CrossRefGoogle Scholar
  14. Majumdar B, Chaudhuri SR, Ray Arun, Bandyopadhyay SK (2002) Potent antiulcerogenic activity of ethanol extract of leaf of Piper betle linn by antioxidative mechanism. Indian J Clin Biochem 17:49–57CrossRefGoogle Scholar
  15. Nalina T, Rahim ZHA (2007) The crude aqueous extract of Piper betle L. and its antibacterial effect towards Streptococcus mutans. Am J Biotechnol Biochem 3:10–15CrossRefGoogle Scholar
  16. Nantitanon W, Yotsawimonwat S, Okonogi S (2010) Factors influencing antioxidant activities and total phenolic content of guava leaf extract. LWT—Food Sci Technol 43:1095–1103Google Scholar
  17. Pietta P (2000) Flavonoids as antioxidants. J Nat Products 63:1035–1042CrossRefGoogle Scholar
  18. Pin KY, Chuah AL, Rashih AA, Mazura MP, Fadzureena J, Vimala S, Rasadah MA (2010) Antioxidant and anti-inflammatory activities of extracts of betel leaves (Piper betle) from solvents with different polarities. J Trop For Sci 22:448–455Google Scholar
  19. Pobłocka-Olech L, Krauze-Baranowska M, Głód D, Kawiak A, Łojkowska E (2010) Chromatographic analysis of simple phenols in some species from the genus Salix. Phytochem Anal 21:463–469CrossRefGoogle Scholar
  20. Prieto P, Pineda M, Aguilar M (1999) Spectrophotometric quantitation of antioxidant capacity through the formation of phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem 269:337–341CrossRefGoogle Scholar
  21. Rajani M, Ravishankara MN, Shrivastava N, Padh H (2001) HPTLC-aided phytochemical fingerprinting analysis as a tool for evaluation of herbal drugs. A case study of Ushaq (Ammoniacum gum). J Planar Chromatogr 14:34–41Google Scholar
  22. Ramji N, Iyer R, Chandrasekaran S (2002) Phenolic antibacterials from Piper betle in the prevention of halitosis. J Ethnopharmacol 83:149–152CrossRefGoogle Scholar
  23. Rathee JS, Patro BS, Mula S, Gamre S, Chattopadhyay S (2006) Antioxidant activity of Piper betle leaf extract and its constituents. J Agric Food Chem 54:9046–9054CrossRefGoogle Scholar
  24. Rawat AKS, Tripathi RD, Khan AJ, Balasubrahmanyam VR (1989) Essential oil components as markers for identification of Piper betle L. cultivars. Biochem Syst Ecol 17:35–38CrossRefGoogle Scholar
  25. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C (1999) Antioxidant activity applying an improved ABTS radical cation decolorizing assay. Free Radic Biol Medicine 26:1231–1237CrossRefGoogle Scholar
  26. Rice-Evans CA, Miller NJ, Bolwell PG, Bramley PM, Pridham JB (1995) The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radic Res 22:375–383CrossRefGoogle Scholar
  27. Rodriguez VMJ, Alberto MR, Manca de Nadra MC (2007) Antibacterial effect of phenolic compounds from different wines. Food Control 18:93–101CrossRefGoogle Scholar
  28. Sakanaka S, Tachibana Y, Okada Y (2005) Preparation and antioxidant of extracts of Japanese persimmon leaf tea (kakinoha-cha). Food Chem 89:569–575CrossRefGoogle Scholar
  29. Santhanam G, Nagarjan S (1990) Wound healing activity of Curcuma aromatica and Piper betle. Fitoterapia 61:458–459Google Scholar
  30. Sarkar M, Gangopadhyay P, Basak B, Chakrabarty K, Banerji J, Adhikary P, Chatterjee A (2000) The reversible antifertility effect of Piper betle Linn. on Swiss albino male mice. Contraception 62:271–274CrossRefGoogle Scholar
  31. Sharma ML, Rawat AKS, Balasubrahmanyam VR, Singh A (1983) Studies on essential oil of betelvine leaf (Piper betle Linn.). Part II. Indian Perfum 27:91–93Google Scholar
  32. Simões-Pires CA, Hmicha B, Marston A, Hostettmann K (2009) A TLC bioautographic method for the detection of α- and β-glucosidase inhibitors in plant extracts. Phytochem Anal 20:511–515CrossRefGoogle Scholar
  33. Singh M, Shakya S, Soni VK, Dangi A, Kumar N, Bhattacharya SM (2009) The n-hexane and chloroform fractions of Piper betle L. trigger different arms of immune responses in BALB/c mice and exhibit anti filarial activity against human lymphatic filarid Brugia malayi. Int Immunopharmacol 9:716–728CrossRefGoogle Scholar
  34. Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic 16:144–158Google Scholar
  35. Sun BS, Ricardo-Da-Silva JM, Spranger MI (1998) Critical factors of vanillin assay for catechins and proanthocyanidins. J Agric Food Chem 46:4267–4274CrossRefGoogle Scholar
  36. Tappayuthpijarn P, Dejatiwongse Q, Pongpech P, Leelaporn A (1982) Antibacterial activity of extracts of Piper betle leaf. Thai J Pharmacol 4:205–212Google Scholar
  37. Wang L, Weller CL (2006) Recent advances in the extraction of nutraceuticals from plant. Trends Food Sci Tech 17:300–312CrossRefGoogle Scholar
  38. Wirotesangthong M, Inagaki N, Tanaka H, Thanakijcharoenpath W, Nagai H (2008) Inhibitory effects of Piper betle on production of allergic mediators by bone marrow-derived mast cells and lung epithelial cells. Int Immunopharmacol 8:453–457CrossRefGoogle Scholar
  39. Young SC, Wang CJ, Lin JJ, Peng PL, Hsu JL, Chou FP (2007) Protection effect of Piper betle leaf extract against carbon tetrachloride-induced liver fibrosis in rats. Arch Toxicol 81:45–55CrossRefGoogle Scholar
  40. Zeng HW, Jiang YY, Cai DG, Bian J, Long K, Chen ZL (1997) Piperbetol, methylpiperbetol, piperol A and piperol B: a new series of highly specific PAF receptor antagonists from Piper betle. Planta Medica 63:296–298CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • H. V. Annegowda
    • 1
  • P. Y. Tan
    • 1
  • M. N. Mordi
    • 1
  • S. Ramanathan
    • 1
  • M. R. Hamdan
    • 1
  • M. H. Sulaiman
    • 1
  • S. M. Mansor
    • 1
  1. 1.Centre for Drug ResearchUniversiti Sains MalaysiaPenangMalaysia

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